Multiple myeloma intra-clonal heterogeneity: evolution and implications of targeted therapy
Intra-clonal heterogeneity is likely to be the essential feature of clonal evolution, disease progression and relapse. Multiple myeloma (MM), B cell malignancy characterized by clonal proliferation of malignant plasma cells in the bone marrow, represents prototypical disease model to study tumor heterogeneity due to high frequency of intra-clonal diversity within malignant clone. MM remains incurable, despite the development of novel therapies. The overall objective of this proposal is to investigate evolution of intra-clonal heterogeneity during the development and progression of MM by combining cellular, molecular, and genetic approaches. Moreover, defining the impact of chemotherapy and/or immunotherapy on intra-clonal selection, together with the role of the tumor microenvironment on clonal dynamics on the level of genetic and cellular complexity, will provide the framework for development of novel personalized diagnostic criteria that will lead to more effective therapeutic strategies. Our emphasize will focus on the process of clonal evolution during the development of MM: from premalignant precursor conditions known as MGUS and smoldering MM without clinical manifestations of diseases but with present cytogenetic and/or gene-expression abnormalities to active disease stages. Moreover, we will evaluate the impact of novel immunomodulatory drugs (lenalidomide and pomalidomide) and proteasome inhibitors (velcade and Ixazomib); and immunotherapy (daratumumab and PD-1/PD-L1 clinical trials) on the dynamic nature of the clonal selection in MM in our ongoing clinical trials, either alone or together with other conventional anti-MM therapies, delineating their ability to prevent recurrence of subclones and resistance to therapy. Furthermore, the role of the tumor microenvironment by characterizing the impact of overall host immunity on clonal selection will be defined. Therefore, a central component of this study are primary patient bone marrow samples from MM patients, especially with high-risk of cytogenetic involvement. Moreover, “metastatic” dissemination process of clinical sequel by circulating tumor cells from peripheral blood of MM patients will also be studied for tracking the clonal evolution of MM and emergence of drug resistance. Taken together, these studies will define the clonal evolution, therapy involved intra-clonal selection, and the role of the tumor microenvironment in clonal architecture in primary patient specimens using: 1. genetic (whole exome/targeting sequencing and gene expression analyses), 2. delineating molecular/signaling pathways such as phosphorylation signatures, transcriptional factors and regulatory molecules, and 3. phenotypic (mass (CyTOF)/flow cytometry analyses) approaches on single cell level. These studies will reveal insights intra-clonal heterogeneity in MM providing the framework for development of novel combination therapeutic strategies targeting clonal heterogeneity and evolution in MM.
Multiple myeloma (MM) represents prototypical disease model to study tumor heterogeneity due to the high frequency of intra-clonal diversity within malignant clone of plasma cells (PC) in the bone marrow.
To better understand the myeloma heterogeneity within complex myeloma pathophysiology, we perform large-scale mass cytometry (CyTOF) analysis in the cohort of bone marrow samples from MM patients (n=188) compared to 10 age-matched healthy donors.
We design a pipeline for deep characterization of PC within the immune ecosystem of the myeloma microenvironment enrolling bone marrow of MGUS, smoldering MM, newly diagnosed and relapsed or relapsed/refractory MM patients.
To evaluate intra and inter-tumor heterogeneity by profiling of PC clusters, we revealed spectrum of PC sub-clonal clusters with phenotypic and signaling variations and abundance supported the idea of sub-clonal heterogeneity within MM tumor.
To further investigate the genetic heterogeneity and clonal evolution characterizing MGUS, smoldering MM, newly diagnosed and relapsed or relapsed/refractory MM patients DNA from 322 and RNA from 144 primary isolated plasma cells of patients diagnosed with MGUS, SMM, NDMM and RRMM were analyzed by targeted NGS (and additionally tested by WES sequencing) and the real-time PCR method using Bio-Rad's Tier 1 and Tier 2 Multiple Myeloma expression arrays, respectively. Moreover, comprehensive analysis of B cell lymphopoiesis reveals various immunophenotyping aberrancies and signaling modulations with frequency assessment in MM patients.
The impact of immune landscape of the tumor microenvironment in MM by high-dimensional approaches revealed significant immune dysfunction that will lead to more effective therapeutic strategies to improve patient outcome. This study will provide the rational for prediction of MM patient status/prognosis and design of targeted therapy in MM on personalized bases.
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This project has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 964264.